In biochemical research and biotechnology, it is often desirable to couple another molecule to a peptide in order to generate a product with novel properties. This involves modifying a peptide in such a manner that it retains its original biospecific function (e.g. the ability to bind to a receptor) but gains, in addition, a new property. Such derivatized peptides may be called peptide conjugates.
Peptide conjugates have a range of current and potential applications. Non-limiting examples include their use in studies of receptor binding and in the isolation of receptors; in biophysical studies of protein three-dimensional structure and mobility; in many types of diagnostic procedures; in the raising of anti-peptide antibodies and in affinity chromatography. Peptide conjugates may also be used in the generation of synthetic enzymes (through the strategic placement of nonpeptide cofactors in peptide chains), and in such direct biomedical applications as tumor imaging and targeted drug delivery. In the latter areas, peptides that are specifically recognized by particular cell types (e.g. malignant tumor cells) may be used as targeting devices that deliver imaging agents or cell-killing drugs to the desired sites in the body.
Peptides are multifunctional organic molecules that possess a range of potentially reactive groups. These include amino groups (one at the amino terminus, others on the side chains of lysyl residues), carboxyl groups (one at the carboxy terminus, others on side chains of glutamyl and aspartyl residues), as well as others including phenol (tyrosine), imidazole (histidine), guanidino (arginine) and indole (tryptophan) groups. An exemplary peptide conjugate formation involves introducing a biotin moiety into a peptide in order to facilitate its detection and quantitation through the strong and specific recognition of biotin by the protein avidin. (Biotin is a small water-soluble vitamin; avidin can be endowed with many properties that facilitate its quantitation, allowing it to serve as the basis of an indirect assay for the biotinylated peptide).
Biotin is an example of a "tagging" group that allows a peptide to be recognized and specifically bound; other groups, known as "reporter groups", may allow information concerning the location and disposition of the peptide to be ascertained directly without the need of an intervening second agent. Biotin is used as an example in the following discussion, but represents any nonpeptide tag or reporter group which might be coupled to a protein or peptide.
Current technology allows the introduction of biotin at a selected type of target site by using a group-specific reagent, i.e. a modified form of biotin designed to react with a selected class of groups on the peptide. For example, in N-hydroxysuccinimidobiotin, biotin is attached to a reactive entity that readily couples to amino groups. The problem with this approach is that the reagent can react with any of the amino groups present in the peptide or protein, giving (in most cases) a mixture of products. One then is left with the unpleasant choice of performing experiments with a heterogeneous preparation that contains a number of different modified peptides, or of fractionating the mixture of products and characterizing each product to identify the site(s) of modification. Each alternative has evident drawbacks. Thus, reagents that are group-specific for target sites normally present in proteins and peptides can give a unique product only in the minority of instances in which the peptide contains just one of the groups to which the reagent is directed.
Another approach has been site-directed modification. As knowledge of protein and peptide structure improves, scientists increasingly understand the relative contributions to biological activity made by particular regions of a protein. For example, it may be known that some part of a protein is unimportant for the biological activity of the protein. If this protein is being conjugated to a non-peptide group, it would be desirable to be able to direct the incoming modifying group to a site in the protein that is not biologically essential. This would greatly increase the probability of the conjugate retaining the native biological properties of the original peptide while also acquiring the new properties conferred by the label. In addition, a single product would be formed, making purification and characterization of the conjugate relatively simple.
Using conventional approaches, such a result could be expected to be achieved only in the minority of cases in which a single group of the type targeted with a group-specific reagent (e.g. an amino group) exists in the region of the peptide selected to receive the modification, while no other group of the same type exists anywhere in the peptide.
Thus, new strategies are required that allow peptides to be modified at unique and preselected locations. A discussion of the need for such technology was given recently in the particular case of biotinylation:
"An effective method has yet to be reported for the selective incorporation of a single biotin molecule into proteins at a predetermined site. The biotin-containing labeling reagents described in . . . this volume are all group specific; such residue-specific biotinylation would therefore be contingent on the presence of a single modifiable group in the desired target protein. Likewise, the selective modification of C- or N-terminal amino acids is also complicated by the presence of aspartic and glutamic acids and lysines in proteins." PA0 "Synthetic peptides play an important role in current biochemical, pharmacological, and immunological research and are widely prepared using solid-phase methodology. After deprotection and cleavage from the solid support, further processing of the peptides is often required, for example, coupling with marker substances (labels) or proteins. Numerous homo- and heterobifunctional-crosslinking reagents have been used for such purposes. However, it is often difficult to achieve selectivity in the coupling reaction since most peptides contain several reactive groups."
A. Schwarz, C. Wandrey, E. A. Bayer, and M. Wilchek (1990) Methods Enzymol. 184, 160-162.
These authors demonstrated an approach that allows biotin to be introduced selectively at the C-terminus of a peptide or protein by an enzymatic procedure.
A second recent discussion of the issue was given by J. W. Drijfhout et al.:
J. W. Drijfhout, W. Bloemhoff, J. T. Poolman and P. Hoogerhout (1990) Anal. Biochem. 187, 349-354.
These authors proposed a solution to the problem in which the N-terminus of synthetic peptides is modified with a group that contains a chemically masked sulfhydryl (--SH) group. At the appropriate time, the --SH group is unmasked and allowed to react with a modifying label which is thereby located at the N-terminus of the peptide.
This information reflects the concern and interest of peptide chemists that a method be defined which will allow a group with useful properties to be added to a peptide at a single, precisely known site. Unlike the methods noted above, such a site should preferably not be limited to the N- or C-terminus of a peptide; ideally, the unique coupling site should be anywhere in a peptide that the chemist chooses. Thus the present invention is directed to a method that meets many of the requirements expressed in the extracts given above from current literature.